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World population is projected to increase well into the next century reaching 9.3 billion by 2050 and with limited arable land available; meeting the growing population's needs is a hard task which requires accelerated progress for cost effective, sustainable yield increases. Arthropod pests are responsible for major damage to the world's important agricultural crops reducing yield and acting as vectors of diseases. Insect attack resistant transgenic crops offer and alternative strategy of pest control compared to a comprehensive reliance upon chemical pesticides. A wide range of transgenic crops which express proteins with insecticidal properties from Bacillus thuringiensis have been commercialised starting from the mid 1990's and have assisted in increasing yields through their ability to kill phytophagous insects, protecting crops and increasing yields. Recently cases of resistance in insect pests to certain strains of B.thuringiensis toxins expressed in transgenic plants have occurred. Resulting in the need to identify novel resistance genes which can be compiled in plants to delay advances in insect resistance to the insecticidal products and widen the domain of pests affected, overall improving the global state of transgenic crops.
The projected increase of the world's 7 billion population rises well into the next century, reaching 9.3 billion by 2050 and a further 10.1 billion by 2100 (UN Population Division 2011). This increase will be primarily visible in developing regions of the world, with Asia staying the most inhabited major world area during the 21st century whereas Africa's population will more than triple with it increasing through 1 billion in 2011 to 3.6 billion in 2100 (Fig. 1). With this population increase comes a greater drive and demand for food and the need for significant increase in food production and productivity to be able to achieve food security.
Feeding a world population in 2050 of 9.1 billion people has been projected to require food production increases or around 70 percent between 2005/07 and 2050. Also developing countries production would be required to almost double (Fig. 2). Adequately feeding the world population would also mean production of foods that are lacking to ensure nutrition security (Bruinsma 2009).
Figure 1. Projected changes in relative population growth, from 1950-2095.
Source: United Nations, Department of Economic and Social Affairs, Population Division (2011). World Population 2010, (Wall Chart).
Figure 2. Targets of cereal production (left). Between 1961 and 2007 cereal production on a global scale has risen from 877 million tonnes to 2351 million tonnes. Rises in production will need to occur to figures of over 4000 million tonnes by 2050 if these forecasted demands are to be reached. Yield increase rate must increase by 37% to meet these demands.
Source: [Based on FAO data] - FAO world agriculture: toward 2030/2050. Interim report, global perspective studies unit (FAO Rome, 2006).
The question is how to tackle this much needed increase. The majority of necessary production increases will arise from large cropping intensity increases and major yield advances with developing countries having to increase these 80 percent in comparison to the production increases from expansion of arable land at around 20 percent in developing countries (Anon 2009).
Closing the Yield Gap - The use of Biotechnology
Major Constraints on productivity arrive from factors such as plant disease, nutrient and land availability, and damage from pests. With the best locally obtained yields depending on the extent of farmers to use and access, things such as water for irrigation, the right seeds and nutrients, and pest management measures. This review will focus specifically on strategies' incorporating transgenic plants to protect crops from insect pest damage.
Of the minimal amount of arthropods which are classified as pests, many of these induce harmfull implications on crops with destruction of world crop production being 14% causeing around "$100 billion of damage each year" (Nicholson 2007). One current measure in place to reduce losses in crop yield due to insect pests is the use of synthetic insecticides which without use, would cause drastic losses in global crop yeild. Insecticides have and are a incredibly effective method for control of crop pests rapidly when crop safety is at risk. However the major limiting factor on the use of insecticides to tackle crop pests is the increasing resistance of insects to insecticides with reports of resistance being reported in more than 500 species (Nicholson 2007). There are also environmental and health concerns around insecticides with improper and unreasonable use of pesticides leading to outbreaks of pests due to the unintentional destruction of the pests natural enemies (Pimentel 2009). Thus other strategies are being investigated to address the global crop pest problem with one of these being the use of recombinant DNA technology.
Recombinant DNA technology is used to produce transgenic crops which have increased stress tolerance, biotic or abiotic. These transgenic crops contribute a significant input into achieving greater food security whether by increasing resistance to disease, insect pests or even by increasing nutrient levels of the plant to enhance the probability of people meeting their dietary needs for a healthy life. Plants containing transgenes are often called transgenic or genetically engineered (GE) crops, however the reality is that all modern crops have been bred and engineered from their wild state by domestication, selection for preferred traits and controlled breeding through time. Currently the major commercialized transgenic crops have undergone simple manipulations to insert genes to benefit the plant for example genes for herbicide tolerance or pest-insect toxin. In the near future developments in combining desirable traits and new novel traits such as resistance to drought in plants will be brought about. However there are issues of public acceptance of biotechnology with differences in acceptance of genetically engineered plants paired to food production in Europe. Currently applications of biotechnology including those of genetic engineering is encouraged but there are some suspicions that application of biotechnological methods towards production of food could jeopardize modern agriculture and the health and safety of our food. However modern molecular biological methods present enormous prospects for expanding production and reducing risks in production of food. Therefore there is a demand for increased acceptance by the public in biotechnology before it can openly assist in improving global food security. This review focuses on insect resistant transgenic crops.
Figure 3. Global Map of Biotech Crop Countries and Mega-Countries in 2011. Source: James, Clive. 2011. Global Status of Commercialized Biotech/GM Crops: 2011. ISAAA Brief No.43. ISAAA: Ithaca, NY.
Insect resistant biotech crops
2011 was the 16th year of commercialisation of biotech crops with a 15 consecutive years of increase and an increase of 12 million ha at a growth rate of 8% from 2010-2011 reaching a record of 160 million ha. Biotech crops have been the fastest adopted crop technology in the history of modern agriculture to date with a 94 times rise in hectarage from 1.7 million in 1996 to 160 million in 2011 (James 2011). Out of all 29 biotech crop planting countries in 2011, 10 were industrial and 19 developing (Fig. 3) with developing countries growing near 50% of the global biotech crops. Adoption of biotech crops by trait sees herbicide resistance having the largest sector with 59% of the global crops compared to 15% of insect resistant trait crops. However it is the stacked gene approach which is the fastest growing area trait wise with 26% of global biotech crop coverage (James 2011). Of insect resistant biotech crops commercialised those expressing Bacillus thuringiensis (Bt) Î´- endotoxins remain the leading and most successful insecticidal toxins
engineered into plants. Positive yield impacts for the use of biotech IR traints in the corn and cotton sectors have occurred in all countries using them (exept genetically modified IR cotton in Australia) (Brookes and Barfoot 2012). The impact that these traits had on yield on average across the area planted from 1996-2010 is +9.96% for corn traits and +14.4% for cotton traits (Brookes and Barfoot 2012) (Fig 4).
Figure 4. Yield impacts on average for the effect of biotech IR traits between 1996-2010 by country and trait. IRCB= resistant to corn boring pests, IRCRW= resistant to corn rootworm. Source: Brookes, G. and P. Barfoot (2012). "The income and production effects of biotech crops globally 1996-2010." GM Crops and Food: Biotechnology in Agriculture and the Food Chain 3(4): 265-272.
The Bt Odyssey
Bacillus thuringiensis is a ubiquitous soil bacterium. The protein crystals it secretes are called Bt-toxins, Î´- endotoxins or crystal (cry) proteins which are insecticidal in nature and are produced within its cells during sporulation. Most strains of the bacterium produce several cry-proteins, each of which shows a rather specific host range (Bravo et al. 2007). An example of this comes from the Cry1A, Cry1Ab and Cry1C genes which code for proteins of the same name which have a specific insecticidal spectrum to larval forms of lepidopteran insect pests for example the codling moth (Cydia pomonella), European corn borer (Ostrinia nubilalis)(Cry1A) or African stem borer (Busseola fusca)(Cry1Ab). Differently the CryA3 protein has an insecticidal spectrum to coleopteran pests an example of which is the Colorado potato beetle (Leptinotarsa decemlineata) (George et al. 2012; Bravo et al. 2007). Plants that expressed Bacillus thuringiensis insecticidal proteins were initially commercialized in the 1996 growing season (Bates 2005), and since then a large variety of crop plants have been genetically engineered so that they exhibit the Î´- endotoxin gene. Engineering of these plants has been undergone to exhibit the active toxin in the plants tissues to the result that insects which feed on the crops are killed by the toxin.
There are two proposed models for the mechanism of action of cry proteins. The first being the pore formation model which is described as follows. On ingestion by susceptible larvae, the crystal proteins dissolve in the alkaline environment of the gut, and the solubilized inactive protoxins are cleaved by midgut proteases yielding 60-70 kDa protease resistant proteins. Toxin activation involves the proteolytic removal of an N-terminal peptide (25-30 amino acids for Cry1 toxins, 58 residues for Cry3A and 49 for Cry2Aa) and approximately half of the remaining protein from the C-terminus in the case of the long Cry protoxins. The activated toxin then binds to specific receptors on the brush border membrane of the midgut epithelium columnar cells before inserting into the membrane. Toxin insertion leads to the formation of lytic pores in microvilli of apical membranes. Subsequently cell lysis and disruption of the midgut epithelium releases the cell contents providing spores a germinating medium leading to a severe septicaemia and insect death (Bravo et al. 2007). The second is a more recently proposed alternative model which is called the signal transduction model. This model states that insect cell death occurs without the formation of pores. The model proposes that the toxicity of Cry proteins is due to activation of Mg+2-dependant signal cascade pathway which is triggered by interaction of monomeric 3d-Cry toxin with the cadherin protein receptor (Soberón et al. 2009). This activates a guanine nucleotide-binding protein which in turn activates an adenylyl cyclase promoting the production of intracellular camp. Increase in camp levels causes protein kinase A activation which in turn activates an intracellular pathway resulting in cell death (Zhang et al. 2006).
Early commercial varieties of insect-resistant transgenic crops expressed single Cry proteins with specific activity against lepidopteran pests such as 'MON 810' which is a variety of genetically modified maize (corn) developed by Monsanto Company and marketed with the trade name YieldGard. It contains a gene from B.thuringiensis (Cry1Ac) that expresses proteins toxic to insects in the order Lepidoptera, such as the European Corn Borer.
More recently released Bt transgenic crop varieties express genes encoding cry proteins and vegetative insecticidal proteins (VIP) active against Coleoptera and Lepidoptera insects, and also sometimes HT genes via gene stacking. A recent commercialised example of this comes from Sygenta and their 'Agrisure® Viptera 3111' trait stack product which includes triple stacked HT traits, protects against 14- above and below ground insects with combination of Both vegetative insecticidal proteins (Vip3A) and cry proteins derived from B.thuringiensis (Sygenta, 2011). By expressing both VIP and Cry proteins, due to the difference in mechanism of action between the two, the durability of the cultivar is effectively extended by decreasing the likelihood of the insects becoming resistant. Xu et al. (1996) demonstrated that expression of CpTI improves rice plant resistance against two species of rice stem borers with them showing significantly increased resistance to the infestation of the two species, the striped stem borer (Chilo suppressalis), and pink stem borer (Sesamia inferens). In 2000 a trait stacked cotton crop expressing Cry1Ac with cowpea trypsin inhibitor (CpTI) was released in China was employed to improve protection representing the only commercial development of a proteinase inhibitor to date (Gatehouse 2011). This is another example of co-expression to reduce likelihood of resistance in insects to the cultivar.
Evolution of the Resistance
Evolution of insect resistance threatens the continued success of transgenic crops producing B.thuringiensis toxins that kill pests (Tabashnik 2008). The first documented case of field evolved resistance to a B.thuringiensis toxin produced by a transgenic crop is of Helicoverpa zea to Cry1Ac in transgenic cotton with the frequency of resistance alleles increasing substantially in some field populations of H. zea (Tabashnik 2008)(Fig 5). Further examples of resistance to B.thuringiensis toxins produced by a transgenic crop come from western corn rootworm (Diabrotica virgifera virgifera) resistance to Cry3Bb1 maize (Gassman 2011). Furthermore field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera) has been reported in northern China (Zhang, 2011). Despite the resistance detected in laboratory bioassays, resistance of these insects to Cry1Ac and Cry3Bb1 expressing cultivars has not caused widespread control failures. These negative effects of resistance to Cry proteins should reduce the use of crops which produce only single toxins and progress towards crops which incorporate two or more B.thuringiensis toxins and other proteins and proteinase inhibitors such as VIP and CpTI.
Figure 5. Resistance in the field from the cumulative planting of Bt crops worldwide from 1996-2007 (>200 million ha). Only detected in 3 lepidopteran species : Helicoverpa zea (bollworm), to Bt cotton producing Cry1Ac), Spodoptera frugiperda (fall armyworm) to Bt corn producing Cry1F, and Busseola fusca (stem borer) to Bt corn producing Cry1Ab. Tabashnik, B. E., et al. (2008). "Insect resistance to Bt crops: evidence versus theory." Nat Biotechnol 26(2): 199-202.
Currently the approaches to tackle resistance include the use of management strategies such as the refuge strategy (involves growing non-Bt crops near the Bt crops) which can slow the evolution of insect resistance by increasing the chances of resistant insects mating with non-resistant insects, resulting in non-resistant offspring. Pyramiding (stacking) of genes encoding different B.thuringiensis toxins or proteinase inhibitors is a strategy that confers greater levels of pest control (Ferry et al. 2004). These proactive countermeasures combined have so far been successful in preventing widespread insect resistance to many insecticidal crops. However from evidence of the evolution of insect resistance to single B.thuringiensis toxin expressing crops in the field it has seemed that the refuge strategy alone, although postpones resistance for many years, is not enough keep evolution of resistance at bay.
By monitoring resistance of insects collected from biotech crop fields and DNA screening and incorporating gene stacking and refuge strategies, it should be possible to stay ahead of the curve within relation to insect resistance. However the search for novel resistance genes needs to continue to identify different genes which can be combined in plants to increase the range of pests affected and to delay the development of insect resistance to the gene products (Peferoen 1997; Ferry et al. 2004).
Future Strategies And Prospects
Potential of insect evolved resistance to transgenic crops is present and although methods to reduce this currently being implimented, alternative strategies to deal with insect pests are being developed. Improved and extended genetic crop resistance is usually seen as a major strategy to prevent agricultural losses due to insects and diseases.
i) Novel insecticidal molecules from bacteria
Future possibilities from bacterial origin includes the toxin complex a (Tca), a high molecular weight insecticidal protein complex produced by the bacterium Photorhabdus luminescens, which has been found to be orally toxic to both the Colorado potato beetle, Leptinotarsa decemlineata, and the sweet potato whitefly, Bemisia tabaci biotype B (Blackburn et al. 2005; Chattopadhyay et al. 2005; ffrench-Constant et al. 2007). Also similar toxins have been described from the taxonomically related Xenorhabdus nematophilus (Morgan et al. 2001; ffrench-Constant et al. 2007). A study by ffrench-Constant et al. (2007) highlighted the fact that both Xenorhabdus and Photorhabdus can penetrate the insect cuticle into the insect haemolymph via their nematode vectors and produce a range of toxins with oral and insecticidal activity. Another future prospective insecticide was discovered in several strains of B.thuringiensis which produce insecticidal proteins during the vegetative phase of growth. These proteins are called vegetative insecticidal proteins (Vip) and do not show any similarity to Î´-endotoxins of B.thuringiensis. VIP's possess toxicity of the same magnitude as that of B.thuringiensis Î´-endotoxins against susceptible insects, with new Vips still being identified (Bhalla et al. 2005).
RNA interference (RNAi) also has considerable potential for the control of pest insects. Baum et al. (2007) and Mao et al. (2007) showed from bioassays of transgenic plants that the development of insects can be delayed and that damage to plants can be reduced by feeding of dsRNA produced by the plant providing promising application purposes for RNAi in insect control.
ii) Plant-derived insecticidal molecules
Of insect resistance genes developed from higher plants there are two major groups used to confer insect resistance on crops: lectins and inhibitors of digestive enzymes (proteinase and amylase inhibitors). The enzyme inhibitors impede digestion through their action on insect gut digestive a-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. Proteinase inhibitors provide plants with a natural endogenous defence system against insects by effecting growth and development of many insects (Murdock and Shade 2002). The digestive system of many insects possesses trypsin and chymotrypsin for digestion and proteinase inhibitors mostly inhibit enzymes by mimicking substrates (Haq et al. 2004). There are different types of proteinase inhibitors with the four discovered called serine, cysteine, aspartic and metallo-protease inhibitors, with cysteine and serine proteinase inhibitors showing the most promising results in combat against Lepidoptera and Coleoptera. Proteinase inhibitors have already been expressed in plants and CpTI has been commercialised but further research could assist in improving crop resistance. Alpha amylase inhibitors from seeds have also been found to play a defensive role and enhance resistance against insect attack (Ishimoto and Chrispeels 1996; Franco et al. 2002). A recent promising study by Dias et al. (2005) showed that expression of an alpha amylase inhibitor gene from rye seeds (Secale cereal) in tobacco plants managed to develop resistance against cotton boll weevil (Anthonomus grandis), providing the grounds for rye inhibitor to be a molecular biology tool to create GM cotton plants with an enhanced resistance to cotton boll weevil.
Lectins are reversible carbohydrate-binding proteins, which can bind to either simple monosaccharides or more complex glycans. (Peumans and Van Damme 1995) and some various lectins have shown some toxic activity against species of the insect orders Homoptera, Coleoptera, Lepidoptera and Diptera (Schuler 1998). However most interest is mainly concentrated on snowdrop lectin, Galanthus nivalis agglutinin (GNA) due to being the first plant lectin shown to be active against Hemiptera. It specifically binds to high-mannose glycans and it has been shown to have a limited negative effect on several aspects of insect life when expressed in plants such as reduced survival, reduced feeding and delayed larval development (Nagadhara et al. 2004; Mehlo et al. 2005). However it is the ability of GNA to transverse the insect gut epithelium and remain stable and active, as it is resistant to proteolytic activity, which allows use of it as a 'carrier' via creation of fusion protein molecules to deliver other peptides to the haemolymph by transporting them across the insect gut (Fitches et al. 2002).
iii) Fusion proteins as insecticidal molecules
Fitches et al. (2004) also demonstrated that GNA can be used to as a carrier protein to deliver an insecticidal spider venom neurotoxin (Segestria florentina toxin 1:SFI1) to the haemolymph of lepidopteran larvae which caused 100% mortality to first stage larvae of tomato moth (Lacanobia oleracea) after six days. Fitches et al. (2012) also demonstrated the ability of GNA to mediate transport of Ï‰-Hexatoxin-Hv1a peptide across the gut epithelium in lepidopteran larva but also the capability of GNA delivering HV1a to sites of action within the insect central nervous system, dramatically enhancing the oral activity of insecticidal proteins. This outlines a potent way to deliver many natural insect selective toxins as novel biopesticides using the huge diversity of toxins from venom of organisms such as spiders, scorpions (Fitches et al. 2010) and sea anemones (Bosmans and Tytgat 2007), possibly providing a whole range of insect specific insecticidal crops.
Pest control in modern agriculture has undergone a true revolution. The increasing moving away from pesticides and the recent advances in genetic engineering has resulted in successful control of many pests of important food crops. The transgenic approach to tackling crop pests should allow increases in yield quality and productivity in an environmentally friendly manner. With the large variety of different novel insecticides possible due to the remarkable diversity of compounds with potential insecticidal activity there should be many different ways to prevent resistance, and if it does occur, the numerous number of compounds with insecticidal activity can be used to engineer new insect resistant crops.